Enhancement of antitumor therapy with hemoglobin-based conjugates

ABSTRACT

The use of hemoglobin-polymer conjugates to enhance antitumor therapy in mammals is disclosed. The methods include administering an effective amount of an antitumor therapy such as radiation in combination with hemoglobin conjugated to a substantially non-antigenic polymer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 07/888,039, now U.S. Pat. No. 5,386,014 filed May 22, 1992,which is a continuation of U.S. patent application Ser. No. 07/440,553,now abandoned. This application is also a Continuation-In-Part of U.S.patent application Ser. No. 07/960,007, filed Oct. 13, 1992, now U.S.Pat. No. 5,312,808. The disclosure of each of these applications isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to enhancing antitumor therapies withhemoglobin-polymer conjugates.

2. Description of Related Art

Solid tumor masses are often hypoxic in mammals. This local hypoxia isbelieved to be at least somewhat responsible for reducing theeffectiveness of current antitumor treatments. In the past, some haveinvestigated whether increasing oxygen inspiration during chemotherapyor radiation could improve tumor kill. While such tactics have intuitiveappeal, it is believed that none have investigated whether increasingoxygen levels directly at the site of the lesion would improve antitumortherapy. The present invention addresses this issue and is describedbelow.

SUMMARY OF THE INVENTION

The present invention provides methods of enhancing the effectiveness ofantitumor treatments such as radiation and/or chemotherapeutic agenttherapies. Methods of reducing tumor burden in mammals are alsoprovided. The methods include administering to a mammal in need of suchtherapy or therapies an effective amount of hemoglobin conjugated to asubstantially non-antigenic polymer in combination with an antitumortherapy. A significant reduction in the tumorous condition results.

The polymer conjugates are preferably in the form of poly(alkyleneoxide)-hemoglobin (PAO-Hb) and most preferably poly(ethyleneglycol)-hemoglobin (PEG-Hb). The conjugates are administered as part ofpharmaceutically-acceptable fluids or solutions as such terms areunderstood in the art. The solutions contain from about 0.2 to about 40wt. % conjugates; preferably about 2-20 wt % and most preferably about10-14 wt % PAO-Hb conjugates. In each case, the conjugates are composedof about 50% Hb.

Suitable hemoglobins for the conjugates include both recombinant,including wild type or mutants, and non-recombinant types whichcorrespond to mammalian species. Human and ruminant hemoglobins such asbovine hemoglobins are preferred.

The amount of PAO-Hb conjugate administered is dependent on severalfactors including tumor type, tumor burden, the amount of radiation andwhether chemotherapeutic agents will also be administered. While thepreference of the artisan will ultimately prevail, the amount of PAO-Hbadministered will range from 0.24-6.30 g/kg.

The Hb-conjugates are preferably administered in combination with a formof radiation therapy. Chemotherapy can also be included. The combinationof therapies principally includes administering the hemoglobinconjugates prior to, or, at the same time as the other antitumortherapies.

The present invention has several advantages over the prior art. It hasunexpectedly been found that the Hb-polymer conjugates preferentiallylocalize in hypoxic areas including tumor lesions. Thus, the hemoglobinconjugates synergistically enhance radiation and/or chemotherapy byproviding high levels of oxygen locally.

For a better understanding of the present invention, reference is madeto the following detailed description, and its scope will be pointed outin the appended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a comparison of average tissue oxygenation in ratosteogenic sarcoma after administration of 6 or 15 ml/kg of eitherPEG-Hb or Ringer's Lactate.

FIG. 2 illustrates a comparison of rat osteogenic sarcoma tumor sizesafter various treatments.

FIG. 3 illustrates the ability of various treatments to achieve completeremission of rat osteogenic sarcoma.

FIG. 4 illustrates a comparison of average tissue oxygenation in rat C6glioma after administration of 6 or 15 ml/kg of either PEG-Hb orRinger's Lactate.

DETAILED DESCRIPTION OF THE INVENTION

A. Introduction

The above-identified related applications as well as U.S. Pat. No.5,234,903, the contents of which are hereby incorporated by reference,disclose various aspects relating to poly(alkylene oxide)-conjugatedhemoglobins. In addition to the utility of PAO-Hb conjugates as atreatment for hypovolemia, shock and anemia, additional research withthe conjugates has now revealed that these compositions unexpectedlyenhance the effectiveness of antitumor treatments.

B. Treatment

The terms "treat" and "treatment" as used herein refer to medicalintervention. The terms relate to repair, reduction, prevention oralleviation of tumorous conditions in mammals. For purposes of thepresent invention, "tumorous condition" and "tumor" are understood toinclude their generally accepted medical meanings. Thus, the termsdescribe physiologic conditions in mammals relating to, or caused by, aneoplastic growth such as that associated abnormal or malignant cellularproliferation.

C. Antitumor Therapy

The invention includes a method of enhancing the effectiveness ofantitumor therapy in mammals. In this aspect, a mammal in need of suchtherapy is administered an effective amount of an antitumor therapy incombination with hemoglobin conjugated to a substantially non-antigenicpolymer.

For purposes of the present invention, antitumor therapy is understoodto mean a therapy specifically designed to combat neoplastic growth. Thetherapy preferably connotes radiation although chemotherapy andcombinations thereof are also within the scope.

The term "radiation" is used in a manner consistent with its customarymedical meaning. Most commonly, X-ray or gamma radiation is administeredusing standard radiation-emitting apparatus. The radiation is directedspecifically at the site of the tumor, although regional or evencomplete body doses can be administered to reduce tumor size. Tumor celldeath is believed to result from the effects of ionization of O₂ into O₂⁻ and/or OH⁻. The conjugates, which preferentially localize in hypoxicareas, thus act synergistically with any radiation therapy which relies,at least in part, on the presence of oxygen for an effect.

The dose of radiation administered can broadly range from 1 to 70 Grays(1 Gy=100 rads). The exact amount given is dependant upon severalfactors including tumor size and location, patient age, physicalcondition and other conditions known to those of ordinary skill in theart. When included as part of the inventive method, the radiation ispreferably given in amounts ranging from 10-50 Gy in the form of asingle or fractionated doses.

The method can also include the administration of chemotherapeuticagents in addition to or in place of radiation. Within this aspect isthe administration of agents known for their specific activity againstneoplastic conditions. A non-limiting list of suitable agents include:

a. alkylating agents such as cyclophosphamide, cis-platin, carboplatin;

b. alkaloids such as vincristine, vinblastine, taxoids and the like; and

c. doxorubicin, methotrexate, bleomycin, misonidazole and others.

It is understood that these agents are given within their known dosageranges when used to treat tumors.

D. Hemoglobin Conjugates

In a preferred embodiment, the conjugates include poly(alkylene oxide)modified hemoglobins such as those disclosed in the parent patentapplications already incorporated by reference herein. In particular,the poly(alkylene oxide) hemoglobin (PAO-Hb) conjugates are preferablyadministered in physiologically-acceptable solutions.

The conjugate is preferably formed by covalently bonding the hydroxylterminals of the poly(alkylene oxide) and the free amino groups oflysine residues of the hemoglobin. See U.S. Pat. No. 5,234,903, whichdiscloses PEG succinimidyl carbonate-Hb. Other art-recognized methods ofconjugating the polymers with the Hb proteins, such as by via an amideor ester linkage, are also suitable for use with the present invention.While epsilon amino group modifications of hemoglobin lysines arepreferred, other conjugation methods are also contemplated. Covalentlinkage by any atom between the hemoglobin and polymer is possible.Moreover, non-covalent conjugation such as lipophilic or hydrophilicinteractions are also contemplated. Moreover, polyethylene glycol-basedliposomes containing either Hb alone or as a PAO-Hb conjugate are alsocontemplated.

Although the preferred conjugates used herein include poly(alkyleneoxides), alternative non-antigenic polymeric substances are also useful.Within this class are materials such as dextran, polyvinyl pyrrolidones,polysaccharides, starches, polyvinyl alcohols, polyacryl amides or othersimilar substantially non-immunogenic polymers. Those of ordinary skillin the art will realize that the foregoing is merely illustrative andnot intended to restrict the type of non-antigenic polymer suitable foruse herein.

The conjugate substituents are typically reacted under conditions whichare appropriate to effect conjugation of the polymer and hemoglobin yetretain the ability of the hemoglobin or hemoglobin-like substance totransfer oxygen. The reactants are combined so that there is aseveral-fold molar excess of the polymeric substance over thehemoglobin. The reactions are carried out at temperatures of from about0° to about 25° C. over time periods ranging from a few minutes to aslong as 12 hours. Following the conjugation reaction, the desiredproduct is recovered using known techniques and purified using columnchromatography or similar apparatus if necessary.

By controlling the molar excess of the polymer reacted with thehemoglobin, the artisan can tailor the number of polymeric strandsattached. Preferable conjugates contain around 11 strands of PEG havebeen made by reacting about a 15 to 20 fold molar excess of an activatedPEG with hemoglobin.

The hemoglobin conjugates preferably have a molecular weight of at leastabout 85,000 daltons and a degree of substitution of at least 5poly(alkylene oxide) conjugates per hemoglobin molecule. Morepreferably, the conjugates have a molecular weight of at least 100,000daltons and at least 8 poly(alkylene oxide) strands per hemoglobinconjugate. Most preferred conjugates have a molecular weight of at least120,000 daltons and at least 11 poly(alkylene oxides) per hemoglobinmolecule.

E. Polymer Portion of Conjugate

The conjugates preferably include polyethylene glycol (PEG) as thepoly(alkylene oxide). The poly(alkylene oxides) includemonomethoxy-polyethylene glycol, polypropylene glycol, block copolymersof polyethylene glycol and polypropylene glycol and the like. Thepolymers can also be distally capped with C₁₋₄ alkyls instead ofmonomethoxy groups.

To be suitable for use herein, the poly(alkylene oxides) must be solublein water at room temperature. Poly(alkylene oxides) having a molecularweight from about 200 to about 20,000 daltons are preferable for useherein, with PAO's having molecular weights of from about 2,000 to about10,000 being preferred and PAO's having a molecular weight of about5,000 being particularly preferred.

F. Hemoglobin Portion

The hemoglobin (Hb) portion of the conjugates can be obtained from anyappropriate mammalian source, human or non-human. Human hemoglobin canbe obtained from whole human blood which has either been freshly drawnor obtained from "out-dated" supplies from blood banks. Human hemoglobincan also be obtained from placentas or packed erythrocytes obtained fromblood donor centers. The hemoglobin can also be obtained fromrecombinant methods including the establishment of transgenic herds orcells. Such transgenic animals express wild type human, variant human,or mutated human hemoglobins, for example. Non-human hemoglobins includeruminant hemoglobins, such as bovine and/or ovine sources. Porcinehemoglobins are also of use. Mammalian-species specific hemoglobins arealso contemplated. Pharmaceutically-acceptable solutions containingmixtures of various types of Hb conjugated to the poly(alkylene oxides)are also contemplated. The hemoglobin portion can account for about20-80 percent of the weight of the conjugates.

G. Hemoglobin-Conjugate Solutions

The amount of conjugates contained in the solution can be in a range ofabout 0.2-40 wt %; solutions containing about 2-20 are preferred andsolutions containing about 10-14 wt% are most preferred. Such solutionsare capable of delivering conjugates having an in vivo half life of atleast 2 hours, preferably, at least 6-18 hours and most preferably atleast 12-20 hours in mammals. Preparations having in vivo half lives ofabout 40-60 hours in mammals are also contemplated. It is to beunderstood that the actual half life of the conjugates in vivo willdepend upon several factors including the species, sex and weight of themammal and dosage administered.

H. Amount of Conjugate Administered

The amount of the conjugate administered is an amount whichsignificantly enhances antitumor therapy. Evidence of enhancement can bededuced by observation or by analytical measurements of increased localmuscle/tissue/organ oxygen levels using apparatus designed for suchpurpose. One such apparatus is an OxySpot/OxyMap available from MedicalSystems of Greenvale, N.Y. The maximum dose is the highest dosage thatdoes not cause clinically important side effects. For purposes of thepresent invention, such side effects in humans include clinicallyimportant hypervolemia, iron overload, renal damage, etc. The conjugatesare usually administered directly into the bloodstream such viaintravenous infusion or transfusion. In the case of transfusionaltherapy, the solutions can be administered in amounts ranging up to 70%of the patient's blood volume. It will be understood that the fluids mayalso contain pharmaceutical necessities such as buffers, preservatives,etc. as such ingredients are utilized in the art.

The amount of the conjugate will depend upon several factors. Forexample, the tumor size, type of tumor, type of radiation and/orchemotherapy as well as the concentration of the conjugated hemoglobinsincluded in the administered solution all will effect the amountadministered. As a general guideline, PAO-Hb conjugates are administeredin amounts ranging from 0.24-6.3 g/kg and preferably in amounts rangingfrom 0.72-2.0 g/kg.

I. Combination Therapy

As used herein, the term "in combination with" is understood to meanthat the Hb-conjugates are administered to the mammal within a timeperiod that allows the conjugates to synergistically interact with theradiation and/or chemotherapeutic agent. In situations where theconjugates are administered in combination with radiation therapy, theconjugates are preferably given at least about 2-4 hours before eachradiation dose. In the case of chemotherapeutic agents, the conjugatesare administered as part of an ongoing polypharmaceutical regimen.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

EXAMPLE I

In this Example, the ability of PEG-Hb conjugates to preferentiallylocalize in osteogenic sarcoma tumor areas was demonstrated using PEG-Hbprepared according as described in U.S. Pat. No. 5,234,903 using bovineHb and contained 12 g % conjugates (approx. 50% Hb), methemoglobin <5%.The OxyMap™ apparatus, a product of Medical Systems, Greenvale, NY, wasused to measure oxygen tension in osteosarcoma in rats. The oxygenmeasurement protocols of Rumsey, W. R. et al in Science 241:1649-51(1988) and Wilson et al in Cancer Res. 52:3988-93 (1992) were followed.

Twelve Sprague-Dawley rats (approx. 200-250 g, female) were eachinjected subcutaneously with approximately 1×10⁷ osteosarcoma cells (0.1ml). The UMR106 tumor cell line was obtained from ATCC. After the tumorshad grown to about 1.5 cm³ in diameter, the rats were anesthetized with20 mg/kg sodium pentobarbital to make a skin incision to expose thetumor. The rats were divided into four groups of three. Two groupsreceived a single bolus IV injection of PEG-Hb while the other groupsreceived single bolus IV injections of Ringer's Lactate. The dosing ofthe rats in each group was as follows:

Group I: Ringer's Lactate @6 ml/kg

Group II: Ringer's Lactate @15 ml/kg

Group III: PEG-Hb @6 ml/kg

Group IV: PEG-Hb @15 ml/kg.

The average baseline tissue oxygen tension in the tumors (n=12) wasdetermined to be about 3.5-4 torr.

RESULTS

A. Group I v. Group III

Referring now to FIG. 1, the effect of a 6 ml/kg bolus injection ofPEG-Hb compared to Ringer's lactate is shown. With PEG-Hb, tissue oxygentension rose immediately from the baseline 3.5-4 torr. to 4.5 torr.After one hour, tissue oxygen tension rose to about 9 torr. Two hoursafter the bolus injection, tissue oxygen tension rose to about 13 torrand remained at about this level for at least 2 more hours before thestudy was ceased. The Ringer's lactate injection failed to provide anynoticeable improvement in local oxygenation.

B. Group II v. Group IV

Similar to the results described above, the effect of a bolus injectionof 15 ml/kg PEG-Hb compared to Ringer's lactate 15 ml/kg is seen. WithPEG-Hb, tissue oxygen tension rose to 15.6 torr two hours afterinjection and remained at about this level for at least two more hours.The Ringer's lactate injection again failed to raise tumor oxygenation.

EXAMPLE II

In this example, the synergy of PEG-Hb with radiation therapy wasdemonstrated in rats. The gamma ray apparatus was a Gamma 40 (AtomicEnergy, Inc.). The PEG-Hb solution was the same as that described inExample 1.

Forty Sprague-Dawley rats(weighing approx. 200-250 g,) were injectedwith a 0.1 ml solution containing approximately 1×10⁷ osteogenic sarcomacells (UMR106, ATCC) in the right hindquarter. The tumors were allowedto grow to about 1.5 cm³ in volume. The rats were divided into fourequal groups of ten.

The first group (A) received 15 ml/kg Ringer's lactate without anyradiation. The second group (B) received 15 ml/kg of Ringer's lactateand a single 4 Gy gamma radiation dose. The third group (C) received 6ml/kg PEG-Hb and a single 4 Gy gamma radiation dose. The final groupreceived 15 ml/kg PEG-Hb and a single 4 Gy gamma radiation dose. Allsolutions were administered via the tail vein. The radiation wasadministered two hours later.

On the day of the experiments, the animals were anesthetized using 20mg/kg of sodium pentobarbital by i.p. injection. The animals were placedon heating pads and their body temperature is maintained at 38-39degrees.

The osteosarcoma was measured prior to the radiation, after one, two,three, and finally, after four weeks. The average results are set forthin FIG. 2. Osteosarcomas larger than 1.5 cm³ are hypoxic, with surfacetissue oxygen tensions of from 0-5 torr, as determined with theOxySpot/OxyMap apparatus,(described above). The value of adding PEG-Hbis dramatically shown in FIG. 2. In both groups C and D virtually nosarcoma was found four weeks after the administration of the conjugatesin combination with the radiation. The Ringer's control groups showedcontinued tumor growth. FIG. 3 shows the rates of complete remission inthe rats given the various treatment methods described herein. Tumorsize was measured externally with calipers. The dramatic synergyachieved by including PEG-Hb with radiation is shown for groups C and D.

EXAMPLE III

In this Example, the ability of PEG-Hb conjugates to preferentiallylocalize in tumor areas was demonstrated using the same type of PEG-Hbconjugates described above in Example I. The OxyMap™ apparatus, aproduct of Medical Systems, Greenvale, N.Y. , was used to measure oxygentension in C6 glioma in rats. The oxygen measurement protocols ofRumsey, W. R. et al in Science 241:1649-51 (1988) and Wilson et al inCancer Res. 52:3988-93 (1992) were followed.

Twelve Wistar rats (150-200 g, female) obtained from Charles RiverLaboratories of NJ were each injected subcutaneously with approximately2×10⁶ C6 glioma cells. Tumor cell line was obtained from ATCC. After thetumors had grown to about 1-2 cm in diameter, the rats were anesthetizedwith 20 mg/kg sodium pentobarbital to make a skin incision to expose thetumor. The rats were divided into four groups of three. Two groupsreceived PEG-Hb while the other groups received Ringer's Lactate. Thedosing of the rats in each group was as follows:

Group I: Ringer's Lactate @6 ml/kg

Group II: Ringer's Lactate @15 ml/kg

Group III: PEG-Hb @6 ml/kg

Group IV: PEG-Hb @15 ml/kg.

The average baseline tissue oxygen tension in the tumors (n=12) wasdetermined to be 3.5-4 torr.

RESULTS

A. Group I v. Group III

Referring now to FIG. 4, the difference between a bolus injection of 6ml/kg PEG-Hb and Ringer's lactate is shown. With PEG-Hb, tissue oxygentension rose from the baseline 3.5-4 torr. to 5.5 torr. after one hourand thereafter to almost 10 torr. The tissue oxygen tension remainedhigh for over 2 hours. The Ringer's lactate injection failed to provideany noticeable improvement in local oxygenation.

B. Group II v. Group IV

In this comparison, the effect of a bolus injection of 15 ml/kg PEG-Hbvs. Ringer's lactate is shown. With PEG-Hb, tissue oxygen tension roseto 11.5 torr two hours after injection and remained at about that levelfor at least two more hours. The Ringer's lactate injection again failedto raise tumor oxygenation.

While there have been described what are presently believed to be thepreferred embodiments of the invention, those skilled in the art willrealize that changes and modifications may be made thereto withoutdeparting from the spirit of the invention. It is intended to claim allsuch changes and modifications that fall within the true scope of theinvention.

What is claimed is:
 1. A method of enhancing the effectiveness ofantitumor therapy in mammals, comprising:administering to a mammal inneed of such therapy an effective amount of radiation in combinationwith hemoglobin covalently conjugated to a poly(alkylene oxide), saidhemoglobin conjugate being present in an amount sufficient to enhancethe effectiveness of said radiation.
 2. The method of claim 1, whereinsaid hemoglobin is selected from the group consisting of recombinant andnon-recombinant hemoglobins.
 3. The method of claim 2, wherein saidhemoglobin is mammalian.
 4. The method of claim 3, wherein saidmammalian hemoglobin is selected from the group consisting of human andruminant hemoglobins.
 5. The method of claim 4, wherein said ruminanthemoglobin comprises bovine hemoglobin.
 6. The method of claim 4,wherein said effective amount of radiation is from about 100-5,000 rads.7. The method of claim 1, wherein said hemoglobin conjugate is in apharmaceutically-acceptable fluid.
 8. The method of claim 7, wherein theconcentration of said hemoglobin conjugate in said fluid is from about0.02 to about 40 weight percent.
 9. The method of claim 8, wherein theconcentration of said hemoglobin conjugate in said fluid is from about 2to about 20 weight percent.
 10. The method of claim 8, wherein theconcentration of said hemoglobin conjugate in said fluid is from about10 to about 14 weight percent.
 11. The method of claim 1, wherein saidpoly(alkylene oxide) comprises a polyethylene glycol.
 12. The method ofclaim 11, wherein said poly(alkylene oxide) is selected from the groupconsisting of monomethoxy(polyethylene glycol), poly(propylene glycol)and block copolymers thereof.
 13. The method of claim 1, wherein saidhemoglobin conjugate has a molecular weight of at least 85,000 daltonsand at least 5 poly(alkylene oxide) conjugates per hemoglobin molecule.14. The method of claim 13, wherein said hemoglobin conjugate has amolecular weight of at least 100,000 daltons and at least 8poly(alkylene oxide) conjugates per hemoglobin molecule.
 15. The methodof claim 14, wherein said hemoglobin conjugate has a molecular weight ofat least 120,000 daltons and at least 11 poly(alkylene oxide) conjugatesper hemoglobin molecule.
 16. The method of claim 1, wherein saidpoly(alkylene oxide) has a molecular weight of about 1,000 to about30,000.
 17. The method of claim 16, wherein said poly(alkylene oxide)has a molecular weight of about 2,000 to about 25,000.
 18. The method ofclaim 17, wherein said poly(alkylene oxide) has a molecular weight ofabout 5,000 daltons.
 19. The method of claim 1, wherein said hemoglobinconjugate has a circulating half-life of at least 2 hours.
 20. Themethod of claim 19, wherein said hemoglobin conjugate has a circulatinghalf-life of at least 10 hours.
 21. The method of claim 20, wherein saidhemoglobin conjugate has a circulating half-life of at least 12 hours.22. The method of claim 1, further comprising administering achemotherapeutic agent with said combination.
 23. The method of claim 1,wherein said hemoglobin conjugate is administered about 2-4 hours beforesaid effective amount of radiation.
 24. A method of reducing tumorburden in mammals, comprising administering to a mammal in need of suchtherapy an effective amount of:(a) radiation, optionally in combinationwith a chemotherapeutic agent in combination with (b) hemoglobincovalently conjugated to a polyalkylene oxide, said hemoglobin conjugatebeing present in amounts sufficient to enhance the effectiveness of saidradiation.
 25. The method of claim 24, wherein said hemoglobin isselected from the group consisting of recombinant and non-recombinanthemoglobins.
 26. The method of claim 24, wherein said hemoglobin ismammalian.
 27. The method of claim 26, wherein said mammalian hemoglobinis selected from the group consisting of human and ruminant hemoglobins.28. The method of claim 27, wherein said ruminant hemoglobin comprisesbovine hemoglobin.
 29. The method of claim 24, wherein said hemoglobinconjugate is administered about 2-4 hours before said effective amountof radiation.